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The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis

Katherine Shim¹, Kimberly J. Blake^2,*, Joseph Jack^2, and Mark A. Krasnow^1,

¹ Howard Hughes Medical Institute and Department of Biochemistry, Stanford University, Stanford, CA 94305-5307, USA
² Department of Anatomy, University of Connecticut Health Center, Farmington, CT 06030, USA
^* Present address: Mitchell College, New London, CT 06320, USA
Present address: 9 Cricket Lane, Simsbury, CT 06070, USA

View larger version (91K): [in a new window]   Fig. 1. Effect of rib mutations on tracheal branch outgrowth. (A) Three tracheal sacs in stage 11 wild-type (+) embryo stained with TL1 antiserum to show apical surface (lumen) of tracheal epithelium. (B) Similar view of stage 13 wild-type embryo. Primary branches have budded and dorsal trunk (DT) branches have fused. (C,D) Stage 13 ribex12 and ribP7 homozygotes. Little branch budding or outgrowth has occurred. (E) Stage 13 bnlP1 homozygote for comparison. (F) Five tracheal metameres of stage 14 wild-type embryo. (G,H) ribex12 and ribP7 homozygotes at similar stage. Outgrowth of most branches is stalled, but there is rare aberrant branch outgrowth (arrowheads). All panels in this and other figures show lateral views (dorsal upwards, anterior leftwards) unless noted. Scale bars: in E, 10 µm in A-E; in H, 10 µm in F-H.

View larger version (73K): [in a new window]   Fig. 2. Basal surface of tracheal epithelium continues to migrate in rib mutants. (A) Budding primary branch growing toward Bnl FGF signaling center. Cytoplasmic processes extend from the basal surface of the lead cells, and cell bodies and apical surface follow. (B,D) Four tracheal metameres (Tr1-4) of stage 12 rib+ (B) and ribex12 (D) embryos stained with TL1 antiserum to show lumen and apical surface. Note limited outgrowth of apical surface in rib mutant. (C,E) Same view of similarly staged rib+ and ribex12 embryos carrying trachealess-lacZ reporter (1-eve-1/+) and immunostained for ß-galactosidase to show tracheal cells and basal surface. Basal cytoplasmic processes extend from the tips of growing branches (brackets) in both rib+ and ribex12 embryos; the processes are sometimes more numerous (asterisks) and longer (arrowhead) in the mutant. (F-K) Confocal fluorescence micrographs of tracheal metamere Tr5 in stage 12 rib+ (F-H) and ribex12 (I-K) embryos carrying trachealess-lacZ (1-eve-1/+) and double stained for the apical marker Crumbs and for ß-galactosidase to show cells and basal surface. Red (Crumbs) channel (F,I), green (ß-galactosidase) channel (G,J), and merged image (H,K). Cytoplasmic processes extend from the tips of growing branches (brackets) in both rib+ and ribex12 embryos. Processes are sometimes more numerous (asterisk) and longer (arrowheads) in the mutant. (L-O) Ventral view (anterior left) of stage 13 rib+ (L,M) and ribex12 (N,O) embryos carrying trachealess-lacZ (1-eve-1/+) and immunostained for ß-galactosidase. Visceral tracheal branches (boxed in L,N) migrate internally onto the gut. Detail of boxed regions (M,O) show two visceral branches on the gut surface; cytoplasmic processes are more numerous in mutant. Scale bars: in D, 20 µm for B-E; in I, 40 µm for F-K; in N, 20 µm for L,N.

View larger version (93K): [in a new window]   Fig. 3. Bnl FGF pathway gene expression and function in rib mutants. (A) In situ hybridization for bnl FGF mRNA (blue) in stage 12 heterozygous rib+ embryo (ribP7/CyO,ftz-lacZ). (Out-of-focus brown staining is immunostaining for ß-galactosidase from ftz-lacZ.) (B) Same view of ribP7 homozygote. bnl mRNA expression pattern is not grossly affected. Arrowheads, bnl expression domains 3', 3, 4, 5/7 (Sutherland et al., 1996). (C) In situ hybridization for btl FGFR mRNA in stage 12 heterozygous rib+ embryo (ribP7/CyO,ftz-lacZ). (D) Same view of ribP7 homozygote. Tracheal branch outgrowth is stalled but btl mRNA is expressed normally. (E) Four tracheal metameres of wild-type embryo double stained for the 2A12 tracheal lumenal antigen (brown) and DSRF/blistered, a Bnl-induced gene (blue). DSRF expression is induced in cells at the ends of primary branches (bracket, arrowheads). (F) Same view of ribex12 embryo. Tracheal branches do not grow out normally and DSRF expression is not induced (asterisks). (G) UAS-bnl/+;69B-GAL4/+ embryo stained for DSRF. Ectopic bnl expression induces DSRF expression throughout the tracheal system. (H) UAS-bnl/+; ribex12; 69B-GAL4/+ embryo stained for DSRF. Although the rib mutation blocks branch outgrowth, it does not prevent bnl induction of DSRF expression throughout the trachea. (To aid in embryo genotyping, this preparation was also stained for ß-galactosidase expressed from a TM3 Ubx-lacZ balancer chromosome in the cross, and hence has higher background staining than G.) (I) UAS-bnl/+; 69B-GAL4/+ embryo stained for diphospho-ERK. Ectopic Bnl expression activates ERK throughout the tracheal system. (J) Same view of UAS-bnl/+; ribex12; 69B-GAL4/+ embryo. Although the rib mutation blocks branch outgrowth, it does not prevent bnl-induced phosphorylation of ERK throughout the trachea. Scale bars: in D, 20 µm for A-D; in F, 10 µm for E,F; in J, 20 µm for G-J.

View larger version (18K): [in a new window]   Fig. 4. Genetic mapping of rib. (A) Map of deficiencies in the 55B-56D region and their lethal complementation behavior with rib (at least three rib alleles were tested). rib maps to the interval (*) between the left endpoint of Df(2R)GC8 (56B5) and the right endpoint of Df(2R)P34 (56C1). (B) Sequence polymorphism mapping of the left endpoint of Df(2R)GC8 and right endpoint of Df(2R)P34. Region deleted (–) or present (+) in Df(2R)GC8 or Df(2R)P34 chromosome as indicated by the absence (–) or presence (+) of a sequence polymorphism at that position (see Materials and Methods). Positions of some known and predicted genes (indicated by CG number) are shown. (C) Meiotic recombination mapping of rib1 with respect to cora2. cora+rib+ recombinant chromosomes (n=42) resulting from a crossover between cora2 and rib1 in a cora2,+/+,rib1 transheterozygote were genotyped for five polymorphic markers (PM1-5) in the region. The percentages of recombinant chromosomes carrying the rib1 chromosome allele of PM1, PM2 and PM3 are shown. None carried the rib1 chromosome allele of PM4 or PM5, which map further to the right (not shown). This mapped rib1 to the right of PM2 (dotted line at 28 kb), in an 9 kb interval (*) bounded by the right endpoint of Df(2R)P34 (dotted line at 37 kb). Extrapolation (broken line) places rib1 in the CG7230 locus. Positions of transcripts in the region are shown. (D) Organization of CG7230 locus and cDNA. Boxes, exons; black, coding region; dotted line, right endpoint of Df(2R)P34; broken line, extrapolated position of rib1 (from C); arrow, actual position of rib1 mutation (see Fig. 5A).

View larger version (45K): [in a new window]   Fig. 5. Primary structure of Rib protein. (A) Diagram showing locations of BTB domain (residues 18-138, gray fill), Pipsqueak (PSQ) motif (367-415, white fill) and bipartite nuclear localization sequence (NLS, 395-412, 409-426). The positions and consequences of ribP7, rib1, rib2, ribz1 and ribP16 mutations are indicated, as are locations of three peptides (P1,P2,P3) used to generate Rib antisera. (B) Sequence alignment of BTB domains of Rib, Longitudinals lacking (Lola), Bric à brac (Bab), Broad-complex (Broad), Tramtrack (Ttk), Pipsqueak (Psq) and Tyrosine kinase related (Tkr). Residues identical to Rib are shaded. Marks below sequence indicate residues identical (*) or similar (.) among proteins shown, or identical among all BTB domain proteins . Large black circle, locations of changes caused by ribP7 mutation and rib2 and ribz1 mutations. (C) Sequence alignment of Psq motifs in Rib, Psq and Tkr. Large black circle, location of ribP16 mutation.

View larger version (123K): [in a new window]   Fig. 6. Expression of rib mRNA. In situ hybridization of wild-type embryos with rib RNA probe at embryo stages indicated. (A) Stage 1 (0-15 minutes after egg lay). No transcript is detected. (B) Stage 5 (3 hours). (C) Stage 7 (3.5 hours). Note expression in cells invaginating to form anterior and posterior midgut (amg, pmg). (D) Dorsal view of same embryo. rib transcript is not detected in cells invaginating to form cephalic furrow (cf) and posterior transverse fold (ptf). (E) Stage 8/9 (4 hours). Expression has turned off in the pmg and initiated in the proctodeum (pr) as it invaginates and in mesoderm as cells take on mesenchymal character and begin migration. (F) Stage 10 (5 hours). rib expression has initiated throughout the epidermis and is enriched in segmental patches (arrowhead). (G) Stage 11 (7 hours), surface view. Note rib expression in tracheal cells as they invaginate to form tracheal pits (tp). (H) Same as G, internal focal plane. Note rib transcript in the amg, pmg, foregut (fg) and hindgut (hg) regions, and mesodermal clusters (ms). (I) Stage 13 (10 hours). rib is expressed throughout the epidermis (ep) as it spreads during dorsal closure. Transcript is also seen in the amg and pmg deep to the focal plane. (J) Stage 13, ventral view. rib transcript is present broadly throughout the embryo including the amg and pmg, as the cells migrate towards each other and form the central gut tube, and the hindgut as it elongates. Broad expression continues in stage 14 and 15 (not shown). Scale bar: 30 µm.

View larger version (96K): [in a new window]   Fig. 7. Rib protein expression and localization. Wild-type (A,C,E) and ribex12 (F) embryos stained with anti-Rib (P3) antiserum Ab21802K. No Rib expression is detected in mutant (F). (B,D) rib RNA distribution at similar stages for comparison. st, stomodeum; ms, mesoderm; pr, proctodeum; ep, epidermis; as, amnioserosa. (G-I) Detail of ventral epidermis in stage 14 embryo double stained with Rib antiserum (fluorescein) and DAPI. (G) DAPI channel. (H) Rib (fluorescein) channel. (I) Merged image. Rib staining is coincident with DAPI except for the centromeric region (arrowhead) which stains intensely only with DAPI. Scale bar: 30 µm in A-F; 2 µm in G-I.

View larger version (16K): [in a new window]   Fig. 8. Schematic of rib tracheal phenotype. In rib mutants, basal cytoplasmic processes extend toward Bnl signaling centers, but cell bodies and apical tracheal surface fail to follow.

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